The goal of this proposal is to validate mutations in the mouse Lrsam1 gene as a model of human inherited peripheral neuropathy, and to determine the cellular pathway in which LRSAM1 protein functions. Heritable sensory and motor neuropathies (HSMNs or Charcot-Marie-Tooth disease, CMT) are a clinically and genetically diverse class of diseases that cause dysfunction and axonal degeneration in the peripheral nervous system. An increasing number of genes underlying axonal neuropathies (type 2 CMTs) are being identified, but the disease mechanisms by which these mutations cause peripheral axon degeneration are not clear. Recently, mutations in LRSAM1 have been identified in both Canadian and Dutch pedigrees presenting with recessive and dominant inherited peripheral axonal neuropathies respectively. LRSAM1 is an E3 ubiquitin ligase with one known substrate, TSG101, which is a component of the ESCRT1 complex and important for late endosome to lysosome vesicular sorting and trafficking, among other functions. Several other Charcot-Marie-Tooth-associated proteins such as SIMPLE, RAB7, and FIG4 are also implicated in endosome/lysosome trafficking. No mammalian experimental model for LRSAM1-associated neuropathy exists, and whether LRSAM1 selectively associates with TSG101 and functions in endosome/lysosome sorting and trafficking in peripheral neurons remain unknown. To address these issues, we propose two aims. In Aim 1, we will use gene trap alleles in mice to produce Lrsam1 mutations and validate these mice as a model of human peripheral neuropathy. We presently have mice derived from two independent insertional mutations. These insertions do result in a loss of Lrsam1 expression and preliminary studies indicate that they cause a peripheral neuropathy phenotype in both heterozygous and homozygous mice, consistent with the human disease. We will continue the molecular, electrophysiological, histological, and behavioral characterization of these mice to establish their validity as a disease model. In Aim 2, we will examine the subcellular localization of LRSAM1 protein to determine if it is associated with other components of the endosome/lysosome trafficking pathway and if it colocalizes with other CMT-associated proteins in this pathway. In addition, we will examine the expression pattern of Lrsam1 in mice to determine if restricted expression may account for the peripheral neuron-specificity of the phenotype. Upon completion of these aims, we will have a new mouse model of human axonal CMT that will be available for preclinical and mechanistic studies. In addition, we will have a better understanding of the cellular pathway in which LRSAM1 functions that will inform future mechanistic studies and possibly illustrate a shared cellular pathway for LRSAM1 and other CMT-associated proteins. PUBLIC HEALTH RELEVANCE: This proposal seeks to validate an animal model of inherited peripheral neuropathy caused by mutations in LRSAM1. Mutations in LRSAM1 have been identified in both Canadian and Dutch pedigrees with inherited axonal neuropathy; however, the disease mechanism, and therefore potential therapeutic strategies, remains unclear. Using mouse resources, we have generated strains of mice lacking Lrsam1 expression. It appears that these mice have a neuropathy comparable to patients, and we will therefore establish these mice as a model for future preclinical and mechanistic studies.